Nuclear receptors are a family of small molecule and hormone-regulated transcription factors that share conserved DNA-binding (DBD) and ligand-binding domains (LBD). Small pharmacological compounds able to bind to the cleft of the ligand-binding domain could alter its conformation and subsequently modify transcription of target genes. Such ligand agonists and/or antagonists have already been successfully designed for 23 nuclear receptors among the 48 previously identified in the human genome (1-3). Of interest, DAX-1 (NR0B1; dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene 1) has been shown to act as a robust transcriptional repressor, inhibiting genes involved in steroidogenesis through interaction with corepressors and regulating the pluripotency of stem cells (4-8). The human DAX-1 gene encodes a protein whose C terminus is similar to the LBD of nuclear hormone receptors, while its N terminus is composed of three cysteine-rich 70 amino acids with little similarity to known proteins (4, 7). Mutations in Dax-1 have been shown to cause the X-linked form of adrenal hypoplasia congenita (AHC), associated with hypogonadotropic hypogonadism (HHG). AHC-HHG-associated mutations share an altered DAX-1 C-terminal domain (5, 9), resulting in loss of transcriptional repression activity (5, 7, 9). This finding suggests that the impairment of the DAX-1 transcriptional activity is directly linked to the pathogenesis of AHC-HHG. In addition, Dax-1 has also been shown to be highly expressed in pediatric Ewing tumors (10). As a result, the identification of selective inhibitors of Dax-1 will serve as useful tools to elucidate the developmental and tumorigenic roles of Dax-1, and its maintenance of stem cell pluripotency.

The purpose of this assay is to determine whether powder samples of compounds identified as Dax-1 inhibitor probe candidates can inhibit activity of the DAX-1 nuclear receptor (NR0B1), a robust transcriptional repressor, as measured by activation of promoter activity of the Dax-1 target gene, Steroidogenic acute regulatory protein (StAR). StAR is a transport protein that regulates cholesterol transfer within the mitochondria, which is the rate-limiting step in the production of steroid hormones. It is primarily present in steroid-producing cells, including theca cells and luteal cells in the ovary, Leydig cells in the testis and cell types in the adrenal cortex.

This cell-based assay employs HEK293 cells co-transfected with a full-length DAX-1 expression construct, an SF-1 expression vector (to activate expression of the StAR reporter), and a luciferase reporter harboring the StAR promoter. As designed, compounds that inhibit DAX-1 activity will reduce the interaction of DAX-1 with corepressors, leading to increased StAR promoter activity and luciferase gene expression, resulting in increased well luminescence. Compounds are tested in triplicate at a single concentration of 100 uM and using a 3-point, 1:2 dilution series, starting at a nominal test concentration of 50 uM.

Protocol Summary:

HEK293 cells were routinely cultured in T-75 flasks containing 15 mL of DMEM (4.5 g/L glucose) medium supplemented with 10% v/v fetal bovine serum and 1% v/v penicillin-streptomycin at 37 C, 5% CO2 and 95% relative humidity (RH). When cells reached about 70% confluency, they were transfected with 1.5 mL of serum-free OptiMEM containing 7.8 ug of the p1.3kb StAR-luc reporter plasmid, 3.9 ug of the pSG.SF-1 expression vector and 3.9 ug of the pSV.DAX-1 expression vector, using 31.25 uL of TransIT-293 transfection reagent. In the absence of a pharmacological positive control, DAX-1 inhibition was mimicked by transfecting cells with the empty vector pSG5 instead of pSV.DAX-1. Four hours post transfection, cells were harvested using 3 mL of trypsin-EDTA solution and resuspended at a concentration of 400,000 cells per mL in the wells of culture-treated white 96-well plates (50 uL per well), diluted in DMEM supplemented as above. Transfected cells were then incubated at 37 C, 5% CO2 and 95% RH for 16 hours before luciferase assay. 50 uL of each dilution of the test compounds or DMSO control to reach the appropriate final concentration were also added to the wells. Transfection experiments were performed in duplicate and repeated three times for each tested drug concentration.

Luciferase assays were performed using the Luciferase Assay System as described by the manufacturer except that 50 uL of Luciferase Assay Reagent were injected in each well using a GloMax luminometer equipped with an automatic injector (Promega). Luminescence in each well was integrated for 10 seconds with a 2-second delay after Luciferase Assay Reagent injection. The relative luciferase activity of each test compound was calculated as follows:

This assay was performed by the assay provider's lab. This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well luminescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided.